Vaccinia virus, a member of the genus Orthopoxvirus in the family of Poxviridae, was used as live vaccine to immunize against the human smallpox disease. Successful world-wide vaccination with vaccinia virus culminated in the eradication of variola virus, the causative agent of the smallpox (The global eradication of smallpox. Final report of the global commission for the certification of smallpox eradication. History of Public Health, No.4, Geneva: World Health Organization, 1980). Since that WHO declaration, vaccination has been universally discontinued except for people at high risk of poxvirus infections (e.g. laboratory workers).
More recently, vaccinia viruses have also been used to engineer viral vectors for recombinant gene expression and for the potential use as recombinant live vaccines (Mackett, M. et al., P.N.A.S. USA, 79:7415-7419 (1982); Smith, G. L et al., Biotech. and Genetic Engineering Reviews 2:383-407, (1984)). This entails DNA sequences (genes) which code for foreign antigens being introduced, with the aid of DNA recombination techniques, into the genome of the vaccinia viruses. If the gene is integrated at a site in the viral DNA which is non-essential for the life cycle of the virus, it is possible for the newly produced recombinant vaccinia virus to be infectious, that is to say able to infect foreign cells and thus to express the integrated DNA sequence (EP Patent Applications No. 83,286 and No. 110,385)). The recombinant vaccinia viruses prepared in this way can be used, on the one hand, as live vaccines for the prophylaxis of infectious diseases, on the other hand, for the preparation of heterologous proteins in eukaryotic cells.
Recombinant vaccinia virus expressing the bacteriophage T7 RNA polymerase gene allowed the establishment of widely applicable expression systems for the synthesis of recombinant proteins in mammalian cells (Moss, B., et al., Nature, 348:91-92 (1990)). In all protocols, recombinant gene expression relies on the synthesis of the T7 RNA polymerase in the cytoplasm of eukaryotic cells. Most popular became a protocol for transient-expression (Fuerst, T. R., et al., Proc. Natl. Acad. Sci. USA, 83:8122-8126 (1986) and U.S. patent application Ser. No. 7,648,971)). First, a foreign gene of interest is inserted into a plasmid under the control of the T7 RNA polymerase promoter. In the following, this plasmid is introduced into the cytoplasm of cells infected with a recombinant vaccinia virus producing T7 RNA polymerase using standard transfection procedures.
This transfection protocol is simple because no new recombinant viruses need to be made and very efficient with greater than 80% of the cells expressing the gene of interest (Elroy-Stein, O. and Moss, B., Proc. Natl. Acad. Sci. USA, 87:6743-6747 (1990)). The advantage of the vaccinia virus/T7 RNA polymerase hybrid system over other transient expression systems is very likely its independence on the transport of plasmids to the cellular nucleus. In the past, the system has been extremely useful for analytical purposes in virology and cell biology (Buonocore, L. and Rose, J. K, Nature, 345:625-628, (1990); Pattnaik, A. K and Wertz, G. W., Proc. Natl. Acad. Sci. USA, 88:1379-1383 (1991); Karschin, A. et al., FEBS Lett. 278: 229-233 (1991), Ho, B. Y. et al., FEBS Lett., 301:303-306 (1992); Buchholz, C. J. et al., Virology, 204:770-776 (1994)). However, important future applications of the vaccinia virus/T7 RNA polymerase hybrid system, as e.g. to generate recombinant proteins or recombinant viral particles for novel therapeutic or prophylactic approaches in humans, might be hindered by the productive replication of the recombinant vaccinia vector.
Vaccinia virus is infectious for humans and upon vaccination during the smallpox eradication campaign occasional serious complications were observed. The best overview about the incidence of complications is given by a national survey in the United States monitoring vaccination of about 12 million people with a vaccine based on the New York City Board of Health strain of vaccinia virus (Lane, J. et al. New Engl. J. Med., 281:1201-1208, (1969)). Therefore the most exciting possibility to use vaccinia virus as vector for the development of recombinant live vaccines has been affected by safety concerns and regulations. Furthermore, most of the recombinant vaccinia viruses described in the literature are based on the Western Reserve strain of vaccinia virus. On the other hand, it is known that this strain has a high neurovirulence and is thus poorly suited for use in humans and animals (Morita et al., Vaccine, 5:65-70 (1987)).
For vector applications health risks would be lessened by the use of a highly attenuated vaccinia virus strain. Several such strains of vaccinia virus were especially developed to avoid undesired side effects of smallpox vaccination. Thus, the modified vaccinia virus Ankara (MVA) has been generated by long-term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A., et al., Infection, 3:6-14 (1975); Swiss Patent No. 568,392)). The MVA virus was deposited in compliance with the requirements of the Budapest Treaty at CNCM (Institut Pasteur, Collection Nationale de Cultures Microorganisms, 25, rue du Docteur Roux, 75724 Paris Cedex 15) on Dec. 15, 1987 under Depositary No. I-721. MVA is distinguished by its great attenuation, that is to say by diminished virulence or infectiosity while maintaining good immunogenicity. The MVA virus has been analyzed to determine alterations in the genome relative to the wild CVA strain. Six major deletions of genomic DNA (deletion I, II, III, IV, V, and VI) totaling 31,000 base pairs have been identified (Meyer, H., et al., J. Gen. Virol. 72:1031-1038 (1991)). The resulting MVA virus became severely host cell restricted to avian cells.
Furthermore, MVA is characterized by its extreme attenuation. When tested in a variety of animal models, MVA was proven to be avirulent even in immunosuppressed animals. More importantly, the excellent properties of the MVA strain have been demonstrated in extensive clinical trials (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167: 375-390 (1987), Stickl et al., Dtsch. med. Wschr. 99: 2386-2392 (1974)). During these studies in over 120,000 humans, including high risk patients, no side effects were associated with the use of MVA vaccine.
MVA replication in human cells was found to be blocked late in infection preventing the assembly to mature infectious virions. Nevertheless, MVA was able to express viral and recombinant genes at high levels even in non-permissive cells and was proposed to serve as an efficient and exceptionally safe gene expression vector (Sutter, G. and Moss, B., Proc. Natl. Acad. Sci. USA 89:10847-10851 (1992)). Recently, novel vaccinia vector systems were established on the basis of MVA, having foreign DNA sequences inserted at the site of deletion III within the MVA genome or within the TK gene (Sutter, G. and Moss, B. Dev. Biol. Stand. Basel, Karger 84:195-200 (1995) and U.S. Pat. No. 5,185,146)).
To further exploit the use of MVA a novel possible way to introduce foreign genes by DNA recombination into the MVA strain of vaccinia virus has been sought. Since the intention was not to alter the genome of the MVA virus, it was necessary to use a method which complied with this requirement. According to the present invention a foreign DNA sequence was recombined into the viral DNA precisely at the site of a naturally occurring deletion in the MVA genome.
The present invention thus, inter alia, comprises the following, alone or in combination:
A recombinant MVA virus containing and capable of expressing at least one foreign gene inserted at the site of a naturally occurring deletion within the MVA genome;
a recombinant MVA virus as above containing and capable of expressing at least one foreign gene inserted at the site of deletion II within the MVA genome;
a recombinant MVA virus as above wherein the foreign gene codes for a marker, a therapeutic gene or an antigenic determinant;
a recombinant MVA virus as above wherein the foreign gene codes for an antigenic determinant from a pathogenic virus, a bacteria, or other microorganism, or from a parasite, or a tumor cell;
a recombinant MVA virus as above wherein the foreign gene codes for an antigenic determinant from Plasmodium Falciparum, Mycobacteria, Herpes virus, influenza virus, hepatitis, or human immunodeficiency viruses.
a recombinant MVA virus as above wherein the antigenic determinant is HIV nef or human tyrosinase;
a recombinant MVA virus as above which is MVA-LAInef or MVA-hTYR;
a recombinant MVA virus as above wherein the foreign gene codes for T7 RNA polymerase;
a recombinant MVA virus as above which is MVA-T7 pol;
a recombinant MVA virus as above wherein the foreign gene is under transcriptional control of the vaccinia virus early/late promoter P7.5;
recombinant MVA viruses as above essentially free from viruses being able to replicate in human cells;
the use of a recombinant MVA virus as above for the transcription of DNA sequences under transcriptional control of a T7 RNA polymerase promoter;
a eukaryotic cell infected by a recombinant MVA virus as any above;
a cell infected by a recombinant MVA virus as above wherein the foreign gene code for T7 RNA polymerase;
a cell infected by a recombinant MVA virus as above wherein the foreign gene code for T7 RNA polymerase, additionally containing one or more expression vectors carrying one or more foreign genes under transcriptional control of a T7 RNA polymerase promoter;
the use of cells as above for the production of the polypeptides encoded by said foreign genes comprising:
a) culturing said cells under suitable conditions, and
b) isolating the polypeptides encoded by said foreign genes.
a cell infected by a recombinant MVA virus as above wherein the foreign gene code for T7 RNA polymerase, additionally containing expression vectors carrying viral genes, and/or a viral vector construct encoding the genome of a viral vector under transcriptional control of a T7 RNA polymerase promoter;
the use of a cells as above for the production viral particles comprising:
a) culturing said cells under suitable conditions, and
b) isolating the viral particles;
a cell infected by a recombinant MVA virus as above wherein the foreign gene code for T7 RNA polymerase, additionally containing
a) an expression vector carrying a retroviral vector construct capable of infecting and directing the expression in target cells of one or more foreign genes carried by said retroviral vector construct, and
b) one or more expression vectors carrying the genes encoding the polypeptides required for the genome of said retroviral vector construct to be packaged under transcriptional control of a T7 RNA polymerase promoter;
the use of cells as above for the production of retroviral particles comprising
a) culturing said cells under suitable conditions, and
b) isolating the retroviral particles;
a vaccine containing a recombinant MVA virus as above wherein the foreign gene code for an antigenic determinant in a physiologically acceptable carrier;
the use of a recombinant MVA virus as above wherein the foreign gene code for an antigenic determinant preparation of a vaccine;
the use of a vaccine as above for the immunization of a living animal body, including a human;
the use of a vaccine as above containing MVA-LAInef for the prevention or treatment of HIV infection or AIDS;
the use of a vaccine as above containing MVA-hTYR for the prevention or treatment of melanomas;
a vaccine comprising as a first component, a recombinant MVA virus as above wherein the foreign gene code for T7 RNA polymerase in a physiologically acceptable carrier, and as a second component a DNA sequence carrying an antigenic determinant under transcriptional control of a T7 RNA polymerase promoter in a physiologically acceptable carrier, the two components being contained together or separate;
the use of a vaccine as above for the immunization of a living animal body, including a human, comprising inoculation of said living animal body, including a human, with the first and second component of the vaccine either simultaneously or with a timelag using the same inoculation site; and
The term xe2x80x9cgenexe2x80x9d means any DNA sequence which codes for a protein or peptide.
The term xe2x80x9cforeign genexe2x80x9d means a gene inserted in a DNA sequence in which it is not normally found.
The foreign gene can be a marker gene, a therapeutic gene, a gene encoding an antigenic determinant, or a viral gene, for example. Such genes are well known in the art.